分离人原代瓣膜细胞进行体外疾病建模
1Division of Cardiology, Department of Medicine, and the Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute,University of Pittsburgh, 2Division of Cardiac Surgery, Department of Cardiothoracic Surgery,University of Pittsburgh and Heart and Vascular Institute, University of Pittsburgh Medical Center, 3Department of Bioengineering,University of Pittsburgh
Summary
该协议描述了在外科主动脉瓣置换手术期间或从尸体组织中提取的人主动脉瓣的收集，以及随后对患者特异性原发性瓣膜内皮和间质细胞的分离、扩增和表征。其中包括有关确保细胞活力和表型特异性所需过程的重要细节。
Abstract
钙化性主动脉瓣疾病（CAVD）存在于近三分之一的老年人口中。主动脉瓣增厚、变硬和钙化会导致主动脉瓣狭窄，并导致心力衰竭和卒中。疾病发病机制是多因素的，炎症、细胞外基质重塑、湍流、机械应力和应变等应激有助于瓣膜内皮细胞和瓣膜间质细胞的成骨分化。然而，驱动健康细胞向钙化细胞成骨转变的精确起始因素尚未完全确定。此外，CAVD诱导的主动脉瓣狭窄目前唯一的治疗方法是主动脉瓣置换术，即切除自体瓣膜（外科主动脉瓣置换术，SAVR）或通过导管插入完全可塌陷的置换瓣膜（经导管主动脉瓣置换术，TAVR）。这些外科手术成本高，风险严重;因此，确定药物发现的新治疗靶点势在必行。为此，本研究开发了一种工作流程，其中手术从患者和供体尸体组织中取出的组织用于创建患者特异性的瓣膜细胞原系，用于体外疾病建模。该协议引入了通常用于器官移植的冷藏解决方案的利用，以减少组织切除和实验室处理之间通常很长的获取时间造成的损害，从而大大稳定了切除组织的细胞。本研究的结果表明，分离的瓣膜细胞在从供体中取出瓣膜后数天以上在培养物中保留其增殖能力以及内皮和间质表型。使用这些材料可以收集对照细胞和CAVD细胞，从中建立对照细胞系和疾病细胞系。
Introduction
钙化性主动脉瓣疾病 （CAVD） 是一种以炎症、纤维化和主动脉瓣叶大钙化为特征的慢性病理学。小叶的进行性重塑和钙化（称为主动脉硬化症）可导致血流受阻（主动脉瓣狭窄），从而导致中风并导致心力衰竭。目前CAVD的唯一治疗方法是手术或经导管主动脉瓣置换术（分别为SAVR和TAVR）。没有非手术选择来阻止或逆转CAVD进展，如果不进行瓣膜置换，死亡率在2-3年内接近50%1，2，3。定义驱动这种病理学的潜在机制将确定潜在的新型治疗方法。
在健康的成人中，主动脉瓣叶的厚度约为1毫米，其主要功能是维持血液单向流出左心室4。三个小叶中的每一个都由一层瓣膜内皮细胞（VEC）组成，该细胞排列在小叶的外表面并起到屏障的作用。VEC通过调节通透性、炎症细胞粘附和旁分泌信号来维持瓣膜稳态5，6，7。瓣膜间质细胞（VIC）包括瓣叶8内的大部分细胞。VIC在传单中排列成三个不同的层次。这些层被称为脑室、海绵和纤维9。脑室面向左心室，含有胶原和弹性蛋白纤维。中间层，海绵状体，含有高蛋白多糖含量，可在心动周期内提供剪切灵活性。外纤维层位于主动脉侧靠近流出面的位置，富含I型和III型原纤维胶原蛋白，可提供在舒张10，11，12期间维持凝结的强度。VIC处于静止状态，然而，炎症，细胞外基质（ECM）重塑和机械应力等因素可能会破坏VIC稳态8，9，13，14，15，16。随着体内平衡的丧失，VIC激活并获得肌成纤维细胞样表型，能够增殖，收缩和分泌重塑细胞外millieu17的蛋白质。活化的VIC可以转变为钙化细胞，这让人联想到间充质干细胞（MSC）分化为成骨细胞15，17，18，19，20，21，22，23，24，25。
钙化似乎在富含胶原蛋白的纤维层中从VEC和VIC的贡献开始，但扩张并侵入小叶的其他层8。因此，很明显，VEC和VICs都对刺激做出反应以上调成骨基因的表达，然而，驱动成骨基因激活的精确事件，以及细胞和小叶细胞外基质之间的复杂相互作用，仍然不清楚。小鼠模型不是研究CAVD发病机制的非遗传驱动因素的理想来源，因为小鼠不会从头发展CAVD26，27，因此使用原代人体组织和从这些组织中分离的原代细胞系是必要的。特别是，随着3D细胞培养和类器官建模领域正在扩大，并且很可能成为鼠模型的离体人类替代品，因此获得大量和高质量的这些细胞势在必行。
本方法的目的是共享一个工作流程，该工作流程已经建立了有效分离和生长从人类供体的手术切除瓣膜中获得的 VEC 和 VIC 的条件。以前的研究表明，从猪28和鼠瓣29中成功分离了VEC和VIC，据我们所知，这是第一个描述这些细胞在人体组织中的分离。这里描述的方案适用于人切除瓣膜，并通过引入冷存储溶液的使用，极大地规避和改善了组织切除和实验室处理之间通常很长的采购时间造成的损害，冷储存溶液是一种临床用于器官移植的缓冲溶液，极大地稳定了切除组织的细胞。此处描述的方案还展示了如何确定细胞表型并保证细胞存活的高效率，同时最大限度地减少细胞交叉污染。
Protocol
所有患者样本均来自匹兹堡大学机构审查委员会根据《赫尔辛基宣言》批准的研究的个体。通过器官恢复和教育中心（CORE）获得的尸体组织得到了匹兹堡大学涉及死者的研究和临床培训监督委员会（CORID）的批准。 1. 批准和安全 根据《赫尔辛基宣言》，获得机构审查委员会 （IRB） 的批准或对任何患者样本或尸体组织收集的豁免备忘录。 参加必要的机构培训?…
Representative Results
上述协议概述了处理人类瓣膜组织以及从这些组织中分离和建立活细胞系所需的步骤。主动脉瓣的小叶被处理用于石蜡包埋，快速冷冻以长期储存以进行生化或遗传分析，并消化以分离VEC和VIC（图1）。虽然手术标本可能具有主动脉瓣狭窄的临床诊断，并且可能表现出肉眼可见的重度钙化结节，但主动脉瓣钙化存在于大量老年人（>65岁）中32，并且由于这种?…
Discussion
从人类获得对照和疾病组织对于体外和离体疾病建模至关重要;然而，虽然人们经常谈到弥合长凳到床边之间的差距的挑战，但相反的顺序 – 从手术室到长凳 – 往往同样令人生畏。对于基础科学家获得原始人体组织标本至关重要的是与投资的外科医生科学家合作，该科学家拥有一支由护士、外科技术人员、医师助理、医学生和住院医师以及临床协议经理组成的团队，他们可以招募和同意患者，参与…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢杰森·多宾斯（Jason Dobbins）对这份手稿的深刻讨论和批判性阅读。我们要感谢器官恢复和教育中心的帮助和支持，并感谢组织捐赠者及其家人使这项研究成为可能。所有患者样本均来自匹兹堡大学机构审查委员会根据《赫尔辛基宣言》批准的研究的个体。通过器官恢复和教育中心（CORE）获得的尸体组织得到了匹兹堡大学涉及死者的研究和临床培训监督委员会（CORID）的批准。
一些用 Biorender.com 创建的图形。
CSH得到了美国国家心肺血液研究所K22 HL117917和R01 HL142932，美国心脏协会20IPA35260111的支持。
Materials
| 0.45 μm filter | Thermo Scientific | 7211345 | Preparing plate with collagen coating |
| 10 cm cell culture plate | Greiner Bio-One | 664160 | Cell culture/cell line expansion |
| 10 mL serological pipet | Fisher | 14955234 | VEC/VIC isolation, cell culture, cell line expansion |
| 1000 μL filter tips | VWR | 76322-154 | Cell culture/cell line expansion |
| 10XL filter tips | VWR | 76322-132 | Cell culture/cell line expansion |
| 15 mL conical tubes | Thermo Scientific | 339650 | Tissue storage, VIC/VEC isolation |
| 16% paraformaldehyde aqueous solution | Electron Microscopy Sciences | 15710S | Tissue and cell fixative |
| 190 proof ethanol | Decon | 2801 | Disinfection |
| 1x DPBS: no calcium, no magnesium | Gibco | 14190250 | Saline solution. VIC/VEC isolation |
| 1x PBS | Fisher | BP2944100 | Saline solution. Tissue preparation, VIC/VEC isolation |
| 20 μL filter tips | VWR | 76322-134 | Cell culture/cell line expansion |
| 200 proof ethanol | Decon | 2701 | Deparaffinizing tissue samples |
| 2-propanol | Fisher | A416P 4 | Making collagen coated plates |
| 5 mL serological pipet | Fisher | 14955233 | VEC/VIC isolation, cell culture, cell line expansion |
| 50 mL conical tubes | Thermo Scientific | 339652 | Tissue storage, VIC/VEC isolation |
| 60 mm dish | GenClone | 25-260 | VEC isolation |
| 6-well cell culture plate | Corning | 3516 | Cell culture/cell line expansion |
| Acetic acid, glacial | Fisher | BP2401 500 | Making collagen coated plates |
| AlexaFluor 488 phalloidin | Invitrogen | A12379 | Fluorescent f-actin counterstain |
| Belzer UW Cold Storage Transplant Solution | Bridge to Life | BUW0011L | Tissue storage solution |
| Bovine Serum Albumin, Fraction V – Fatty Acid Free 25g | Bioworld | 220700233 | VEC confirmation with CD31+ Dynabeads |
| Calponin 1 antibody | Abcam | ab46794 | Primary antibody (VIC positive stain) |
| CD31 (PECAM-1) (89C2) | Cell Signaling | 3528 | Primary antibody (VEC positive stain) |
| CD31+ Dynabeads | Invitrogen | 11155D | VEC confirmation with CD31+ Dynabeads |
| CDH5 | Cell Signaling | 2500 | Primary antibody (VEC positive stain) |
| Cell strainer with 0.70 μm pores | Corning | 431751 | VIC isolation |
| Collagen 1, rat tail protein | Gibco | A1048301 | Making collagen coated plates |
| Collagenase II | Worthington Biochemical Corporation | LS004176 | Tissue digestion. Tissue preparation, VIC/VEC isolation |
| Conflikt Ready-to-use Disinfectant Spray | Decon | 4101 | Disinfection |
| Countess II Automated Cell Counter | Invitrogen | A27977 | Automated cell counter |
| Countess II reusable slide coverslips | Invitrogen | 2026h | Automated cell counter required slide cover |
| Coverslips | Fisher | 125485E | Mounting valve samples |
| Cryogenic vials | Olympus Plastics | 24-202 | Freezing cells/tissue samples |
| Disinfecting Bleach with CLOROMAX – Concentrated Formula | Clorox | N/A | Disinfection |
| DMEM | Gibco | 10569044 | Growth media. VIC expansion |
| EBM – Endothelial Cell Medium, Basal Medium, Phenol Red free 500 | Lonza Walkersville | CC3129 | Growth media. VEC expansion |
| EGM-2 Endothelial Cell Medium-2 – 1 kit SingleQuot Kit | Lonza Walkersville | CC4176 | Growth media supplement. VEC expansion |
| EVOS FL Microscope | Life Technologies | Model Number: AME3300 | Fluorescent imaging |
| EVOS XL Microscope | Life Technologies | AMEX1000 | Visualizing cells during cell line expansion |
| Fetal Bovine Serum – Premium Select | R&D Systems | S11550 | VIC expansion |
| Fine scissors | Fine Science Tools | 14088-10 | Tissue preparation, VIC/VEC isolation |
| Fisherbrand Cell Scrapers | Fisher | 08-100-241 | VIC expansion |
| Fungizone | Gibco | 15290-026 | Antifungal: Tissue preparation, VIC/VEC isolation |
| Gentamicin | Gibco | 15710-064 | Antibiotic: Tissue preparation, VIC/VEC isolation |
| Glass slides | Globe Scientific Inc | 1358L | mounting valve samples |
| Goat anti-Mouse 488 | Invitrogen | A11001 | Fluorescent secondary Antibody |
| Goat anti-Mouse 594 | Invitrogen | A11005 | Fluorescent secondary Antibody |
| Goat anti-Rabbit 488 | Invitrogen | A11008 | Fluorescent secondary Antibody |
| Goat anti-Rabbit 594 | Invitrogen | A11012 | Fluorescent secondary Antibody |
| Invitrogen Countess II FL Reusable Slide | Invitrogen | A25750 | Automated cell counter required slide |
| Invitrogen NucBlue Fixed Cell ReadyProbes Reagent (DAPI) | Invitrogen | R37606 | Fluorescent nucleus counterstain |
| LM-HyCryo-STEM – 2X Cryopreservation media for stem cells | HyClone Laboratories, Inc. | SR30002 | Frozen cell storage |
| Mounting Medium | Fisher Chemical Permount | SP15-100 | Mounting valve samples |
| Mr. Frosty freezing container | Nalgene | 51000001 | Container for controlled sample freezing |
| Mycoplasma-ExS Spray | PromoCell | PK-CC91-5051 | Disinfection |
| Penicillin-Streptomycin | Gibco | 15140163 | Antibiotic. VIC expansion |
| Plasmocin | Invivogen | ANTMPT | Anti-mycoplasma. VIC/VEC isolation and expansion |
| SM22a antibody | Abcam | ab14106 | Primary antibody (VIC positive stain) |
| Sstandard pattern scissors | Fine Science Tools | 14001-14 | Tissue preparation, VIC/VEC isolation |
| Sterile cotton swab | Puritan | 25806 10WC | VEC isolation |
| Swingsette human tissue cassette | Simport Scientific | M515-2 | Tissue embedding container |
| Taylor Forceps (17cm) | Fine Science Tools | 11016-17 | Tissue preparation, VIC/VEC isolation |
| Trypan Blue Solution, 0.4% | Gibco | 15250061 | cell counting solution |
| TrypLE Express Enzyme | Gibco | 12604021 | Splitting VIC/VECs |
| Von Kossa kit | Polysciences | 246331 | Staining paraffin sections of tissues for calcification |
| von Willebrand factor antibody | Abcam | ab68545 | Primary antibody (VEC positive stain) |
| Xylenes | Fisher Chemical | X3S-4 | Deparaffinizing tissue samples |
| αSMA antibody | Abcam | ab7817 | Primary antibody (VIC positive stain) |
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Citer Cet Article
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